Iskanje po katalogu digitalne knjižnice

Opis: Subtle physiological patterns within the human organism, such as molecular fluctuations or systemic adaptations, often remain hidden from direct observation due to the inherent variability and noise within biological samples. The liver, a vital organ essential for systemic regulation and toxicological assessment, presents this challenge due to its heightened activity, which can influence enzyme dynamics and metabolic processes. Unlike direct observation, in vitro liver models offer a more precise means of understanding these trends, providing insights into the organ's (patho)physiology, and serving as a platform for toxicity evaluation. However, current liver models lack essential features required to faithfully replicate the liver's microenvironment, resulting in reduced accuracy in toxicity assessments. Furthermore, while researchers emphasize mechanistic insights, such as the molecular processes governing glucose metabolism and cellular energy production, clinicians focus on tangible health outcomes, like blood glucose levels in patients. This disconnect between the objectives and methodologies of basic researchers and clinicians amplifies this gap, hindering effective translational research. Addressing these challenges, a novel liver cell culture system that resembles the in vivo liver microenvironment with clinical instrumentation has been proposed to enhance current liver models, improving their capacity to emulate in vivo conditions. This study introduces a novel liver cell culture system, utilizing a 96-well plate format incorporating hepatic sinusoidal endothelial cells, hepatic stellate cells, Kupffer cells, and hepatocytes to replicate the liver microenvironment. The model integrates clinical instrumentation, specifically a biochemical analyzer to ensure biomarkers closely align with those observed in clinical diagnostics. This design enables researchers to fine-tune conditions that mimic the liver's microanatomy and physiological responses, enhancing its translational potential for toxicity assessments. The approach involves primary cell culture preparation, supernatant analysis through a clinical biochemistry analyzer, and cell viability assessment using the Membrane Potential Cell Viability Assay (MPCVA) method. Additionally, advanced imaging techniques and data analysis tools are incorporated to refine the model's capabilities and ensure greater translatability to clinical applications.
Ključne besede: in vitro toxicity, liver (patho)physiology, liver in vitro model, membrane potential cell viability assay, translational research
Objavljeno v DKUM: 14.08.2025; Ogledov: 0; Prenosov: 0
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Opis: Humans are frequently exposed to environmental hepatotoxins, which can lead to liver failure. Biosensors may be the best candidate for the detection of hepatotoxins because of their high sensitivity and specificity, convenience, time-saving, low cost, and extremely low detection limit. To investigate suitability of HepG2 cells for biosensor use, different methods of adhesion on stainless steel surfaces were investigated, with three groups of experiments performed in vitro. Cytotoxicity assays, which include the resazurin assay, the neutral red assay (NR), and the Coomassie Brilliant Blue (CBB) assay, were used to determine the viability of HepG2 cells exposed to various concentrations of aflatoxin B1 (AFB1) and isoniazid (INH) in parallel. The viability of the HepG2 cells on the stainless steel surface was quantitatively and qualitatively examined with different microscopy techniques. A simple cell-based electrochemical biosensor was developed by evaluating the viability of the HepG2 cells on the stainless steel surface when exposed to various concentrations of AFB1 and INH by using electrochemical impedance spectroscopy (EIS). The results showed that HepG2 cells can adhere to the metal surface and could be used as part of the biosensor to determine simple hepatotoxic samples.
Ključne besede: HepG2 cell line, impedance biosensor, adhesion, hepatotoxins, stainless steel
Objavljeno v DKUM: 10.04.2025; Ogledov: 0; Prenosov: 10
Celotno besedilo (1,87 MB)
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Opis: Over the past century, numerous methods for assessing cell viability have been developed, and there are many different ways to categorize these methods accordingly. We have chosen to use the Organisation for Economic Co-operation and Development (OECD) classification due to its regulatory importance. The OECD categorizes these methods into four groups: non-invasive cell structure damage, invasive cell structure damage, cell growth, and cellular metabolism. Despite the variety of cell viability methods available, they can all be categorized within these four groups, except for two novel methods based on the cell membrane potential, which we added to the list. Each method operates on different principles and has its own advantages and disadvantages, making it essential for researchers to choose the method that best fits their experimental design. This review aims to assist researchers in making this decision by describing these methods regarding their potential use and providing direct references to the cell viability assessment methods. Additionally, we use the OECD classification to facilitate potential regulatory use and to highlight the need for adding a new category to their list.
Ključne besede: cell viability, cell-based methods, in vitro toxicology, OECD cell viability classification
Objavljeno v DKUM: 13.02.2025; Ogledov: 0; Prenosov: 8
Celotno besedilo (2,77 MB)
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Opis: Background : Campylobacter spp. are a major cause of bacterial food-borne diarrhoeal disease. This mainly arises through contamination of meat products during processing. For infection, Campylobacter spp. must adhere to epithelial cells of the mucus layer, survive conditions of the gastrointestinal tract, and colonise the intestine of the host. Addition of probiotic bacteria might promote competitive a dhesion to epithelial cells, consequently reducing Campylobacter jejuni colonisation. Effect of Lactobacillus spp. (PCS20, PCS22, PCS25, LGG, PCK9) on C. jejuni adhesion, invasion and translocation in pig (PSI cl.1) and chicken (B1OXI) small-intestine cell lines, as well as pig enterocytes (CLAB) was investigated. Results : Overall, in competitive adhesion assays with PSI cl.1 and CLAB cell monolayers, the addition of Lactobacillus spp. reduced C. jejuni adherence to the cell surface, and negatively affected the C. jejuni invasion. Interestingly, Lactobacillus spp. significantly impaired C. jejuni adhesion in three-dimensional functional PSI cl.1 and B1OXI cell models. Also, C. jejuni did not translocate across PSI cl.1 and B1OXI cell monolayers when co-incubated with probiotics. Among selected probiotics, Lactobacillus rhamnosus LGG was the strain that reduced adhesion efficacy of C. jejuni most significantly under co-culture conditions. Conclusion : The addition of Lactobacillus spp. to feed additives in livestock nutrition might be an effective novel strategy that targets Campylobacter adhesion to epithelial cells, and thus prevents colonisation, reduces the transmission, and finally lowers the incidence of human campylobacteriosis.
Ključne besede: Campylobacter jejuni, Lactobacillus spp., chicken and pig cell line, adhesion, invasion, translocation
Objavljeno v DKUM: 06.02.2025; Ogledov: 0; Prenosov: 7
Celotno besedilo (1,80 MB)
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Opis: Background: The crucial steps in beta cell stimulus-secretion coupling upon stimulation with glucose are oscillatory changes in metabolism, membrane potential, intracellular calcium concentration, and exocytosis. The changes in membrane potential consist of bursts of spikes, with silent phases between them being dominated by membrane repolarization and absence of spikes. Assessing intra- and intercellular coupling at the multicellular level is possible with ever-increasing detail, but our current ability to simultaneously resolve spikes from many beta cells remains limited to double-impalement electrophysiological recordings. Methods: Since multicellular calcium imaging of spikes would enable a better understanding of coupling between changes in membrane potential and calcium concentration in beta cell collectives, we set out to design an appropriate methodological approach. Results: Combining the acute tissue slice method with ultrafast calcium imaging, we were able to resolve and quantify individual spikes within bursts at a temporal resolution of >150 Hz over prolonged periods, as well as describe their glucose-dependent properties. In addition, by simultaneous patch-clamp recordings we were able to show that calcium spikes closely follow membrane potential changes. Both bursts and spikes coordinate across islets in the form of intercellular waves, with bursts typically displaying global and spikes more local patterns. Conclusions: This method and the associated findings provide additional insight into the complex signaling within beta cell networks. Once extended to tissue from diabetic animals and human donors, this approach could help us better understand the mechanistic basis of diabetes and find new molecular targets.
Ključne besede: beta cell, calcium imaging, calcium oscillations, calcium spikes, physiology
Objavljeno v DKUM: 24.01.2025; Ogledov: 0; Prenosov: 7
Celotno besedilo (9,70 MB)
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